Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525
The Cuicular Hydrocarbons Proiles in the Colonial Recogniion of the Neotropical Eusocial
Wasp,
Mischocytarus cassununga
(Hymenoptera: Vespidae)
Introduction
Cuticular hydrocarbons (CHCs) are present on the surface of all insects and play an important role in the life of insects. These cuticular lipids have evolved for prevention of desiccation, as a barrier to microorganisms and as a important signals in the recognition system of social insects (Hadley, 1981; Lockey, 1988; Singer, 1998; Howard & Blomquist, 2005). Each individual of a colony presents a blend of compounds on its cuticle that may carry information regarding its sex, age, caste, group task as well as colony (Howard & Blomquist, 2005; Provost et al., 2008). These chemical messengers are determined by genetic (Ratnieks, 1991; Page et al., 1991; Arnold et al., 1996) and environmental components (Ratnieks, 1991; Gamboa, 1996).
Based on differences of cuticular compounds, individuals from the same colony are able to detect speciic chemical Abstract
Cuicular hydrocarbons arechemical messengers with fundamental role in informaion transfer on the nestmate recogniion, physical or behavioral caste, age, task specializaion and reproducive status among individuals of colony. In basal (primiively) eusocial wasps, the role of cuicular hydrocarbons in the colonial recogniion has been extensively studied in genus Polistes (Polisinae) and Stenogastrinae. Although the genus Mischocytarus (Polisinae) is a large group of neotropical eusocial wasps and its cuicular hydrocarbons were only invesigated in a few species. This study aimed to verify whether the cuicular hydrocarbons can discriminate the intercolonial idenity in Mischocytarus cassununga. Our results showed that the cuicular hydrocarbons potenially can play a role in intercolony recogniion.
Sociobiology
An international journal on social insects
ASN Murakami1, TM Nunes2, IC Desuó1, SN Shima1, S Mateus2
Aricle History
Edited by
Fabio S. do Nascimento, USP, Brazil Received 01 September 2014 Iniial acceptance 23 October 2014 Final acceptance 10 January 2015
Keywords
Recogniion nestmate, Chemical signal, Colony, Mischocytarini.
Corresponding author
André Sunao Nishiuchi Murakami
Dep. de Zoologia, Insituto de Biociências, Universidade Estadual Paulista
Campus de Rio Claro Av. 24-A, n. 1515, Bela Vista CEP: 13506-900, Rio Claro- SP, Brazil E-Mail: sunamigobio@yahoo.com.br
signals in order to recognize the caste to which they belong as well as identify individually the nestmates, and differentiate the invaders (Gamboa, 1996; Lorenzi et al., 1997; Singer & Espelie, 1997; Singer, 1998; Howard & Blomquist, 2005; Richard & Hunt, 2013). Studies on chemical signaling have shown that the proiles of cuticular hydrocarbons can be used in the recognition among nestmates due to its colony-speciic composition in ants (Bonavita-Cougourdan et al., 1987; Vander Meer et al., 1989; Wagner et al., 2000; Sturgis & Gordon, 2011; Wilgenburg et al., 2011), bees (Breed & Stiller, 1992; Breed et al., 1995; Breed et al., 1998; Dani et al., 2005; Nunes et al, 2008; Nunes et al, 2009; Nunes et al., 2011), wasps (Pfenning et al., 1983; Bonavita-Courgoudan et al., 1991; Gamboa et al., 1996; Dani et al., 2001; Tannure-Nascimento et al., 2007; Bruschini et al., 2011; Mitra et al., 2014) and termites (Haverty & Thorne, 1989; Bagnères et al., 1991; Kaib et al., 2002; Kaib et al., 2004. RESEARCH ARTICLE - WASPS
ASN Murakami et al - Cuticular Hydrocarbons Proiles and Colonial Recognition in Mischocyttarus cassununga 110
In independent founding wasps, cuticular hydrocarbons have been mainly studied in the subfamilies Polistinae (in Polistes) and Stenogastrinae. On the other hand, few studies have investigated the chemical communication in genus Ropalidia and Mischocyttarus. Studies show that the mechanism of recognition of conspeciics depends mainly on the amount of relative concentration of cuticular compounds in a colony. The colony - speciic composition, where females of a given colony are able to recognize their conspeciics by odour, was demonstrated in Polistes annularis (Espelie & Hermann, 1990) , in P. metricus (Espelie et al., 1990; Layton et al., 1994) , in P. dominulus (Bonavita-Courgoudan et al., 1991; Dani et al., 2001; Pickett et al., 2000; Lorenzi et al., 2004; Sumana et al., 2005) , in P. fuscatus (Gamboa et al., 1996; Panek et al., 2001; Pfenning et al., 1983) in P. biglumis bimaculatus (Lorenzi et al., 1997) , and P. satan (Tannure-Nascimento et al., 2007). In subfamily Stenogastrinae, individual recognition through a similar or homogeneous colony odour was also demonstrated in Liostenogaster lavolineata (Cervo et al., 1996; Cervo et al., 2002), in L. vechti (Cervo et al., 1996) Parischnogaster jacobsoni (Cervo et al., 1996) and Parischnogaster striatula (Zanetti et al., 2001). And more recently, in Ropalidia marginata has also demonstrated the importance of colonial proile of cuticular hydrocarbons (Mitra et al., 2014).
The tribe Mischocyttarini is a large group of eusocial wasps with about 245 described species (Carpenter, 1993; Silveira, 2008), however the cuticular hydrocarbons and their role in chemical communication of this group of social insects were investigated in a few species (Ferreira et al., 2012; Neves et al., 2012). Mischocyttarus cassununga is a species of neotropical wasp characterized by a linear and well stable dominance hierarchy, high longevity and rare replacement of the reproductive dominant female (“queen”) in the colony(Murakami & Shima, 2009; Murakami & Shima, 2010). The aims of the present study were to verify whether intercolonial identity could be discriminated using the cuticular hydrocarbons.
Material and Methods
The research was realized at Universidade Estadual Paulista - UNESP, Campus of Rio Claro - SP, using eight (8) colonies of M. cassununga, during the post-emergence phase. All colonies were collected from the same population.
Chemical analysis of the colonies
After the collection of colonies, all female wasps were placed separately in glass vial (1.5 ml) in freezer (temp. ca. -20oC) until the moment of the extraction of the cuticular compounds. The cuticular compounds of each sample were extracted by hexane (Labsynth) at 500 ml for 1 minute. Each sample extract was analysed by gas chromatography coupled with mass spectrometry (SHIMADZU, model QP2010, Tokyo, Japan), equipped with a ZB-5MS column; length: 30 m; ID: 0.25 mm; ilm thickness: 0.25 μm. The column was initially
set at 150 oC for 3 min, then programmed at a rate of 3 oC/min to 280 oC, and then held at 280 oC for 15 min. The carrier gas was helium at a low rate of 1 ml/min. The injection mode was splitless and the injection temperature was 250 oC. For the identiication of the cuticular compounds it was performed comparisons of mass spectra with the Wiley library and with the data of the diagnostic ions present in the literature. All the process of the hydrocarbons identiication was carried out with the aid of the program GCMS solutions for Windows (Shimadzu Corporation).
Statistical analysis
Statistical analyses were conducted in Statistica 7.0 for Windows (StatSoft Inc., Tulsa, OK, U.S.A.). The concentration of all compounds was transformed according to Reyment’s formula (Z = ln (Ap / g (Ap) )), where Ap is the peak area, g (Ap) is the geometric mean of the peak in each group of females and Z is the transformed area of the peak (AITCHISON, 1986). Compounds that, on average, contributed less than 1% to the overall chemical proile (i.e. linear alkanes, monomethyl and dimethyl alkanes) were excluded from the analysis, as made by Kather et al. (2011). The areas of these peaks were recalculated, taking into account the reduced data set, and then reanalysed by statistical tests. First, a one-way ANOVA was carried out to test for group differences (colonies) in total compound quantity for each chemical compound. At the same time, a two-way MANOVA with Pillai test and post-hoc Tukey test was used to test for group differences for each chemical compound separately.
Results and Discussion
The analysis of cuticular compounds of Mischocyttarus cassununga resulted in a total of 88 peaks detected, divided into linear alkanes, methyl-branched alkanes and dimethyl-branched alkanes, with chains between 16 and 36 carbon atoms. The selected peaks for chemical analysis are indicated in igure 1. The overall composition of hydrocarbons on an insect’s cuticle, termed as the proile, can range from highly complex to relatively simple (Dani et al., 2001). For example, in the genus Polistes, the most studied group of basal eusocial wasp, the species Polistes biglumis bimaculatus (Lorenzi et al., 1997) and P. dominulus (Dapporto et al., 2004) have respectively 63 and 65 cuticular compounds, while P. metricus (Layton et al., 1994) and P. satan (Tannure-Nascimento et al., 2007) have cuticular compounds 25 and 28, respectively.
1996; Dani et al., 2001; Dani et al., 2005). In honeybees, the alkenes or unsaturated fatty acids seem to inluence more in the colonial recognition than the alkanes and unsaturated fatty acids (Breed, 1998; Breed et al., 1998). Dani et al. (2005) similarly showed that linear alkanes have no effect on recognition in honeybee, in which only branched alkanes and alkenes are found. According to a comparative study of cuticular hydrocarbons of 78 species of 5 subfamilies of ants, the wide diversity of dimethylalkanes can indicate them as excellent compounds discriminatory recognition inter- and intra-speciic species (Martin & Drijfhout, 2009). Dani et al. (2001) tested the effect of the concentration of some cuticular substances during the process of recognition between conspeciic of Polistes dominulus (Vespidae), and found that linear alkanes did not cause any effect on the recognition response, unlike methylalkanes and alkenes. In the same species (P. dominulus), Lorenzi et al. (2004) found that total quantities of CHCs increased after emergence, with branched alkanes increasing drastically when compared with other classes of hydrocarbons. Older wasps did not incorporate hydrocarbons, suggesting that the chemical proiles of mature wasps are less prone to chemical shifts than those of newly emerged wasps. In stingless bees, newly emerged workers have a very simple hydrocarbon pattern on their cuticle consisting almost entirely of linear alkanes (mainly C23; C25; C27 and C29) and guards were not able to discriminate between newly emerged nestmates and non-nestmates (Nunes et al., 2011). In M. cassununga, all colonies signiicantly differed according to total quantity of fourteen cuticular hydrocarbons (=eight peaks) (Table 1 and Figure 2). The branched alkanes were the predominant compounds in the chemical differentiation of individuals. Differently, in P. satan (Vespidae), the linear alkanes were the main compounds responsibles by the differentiation of individuals through the colonial proile (Tannure-Nascimento et al., 2007; 2008).
Fig 1. Total ion mass chromatograms of a Mischocyttarus cassununga
female wasp. Peak numbers from 1 to 18 correspond to the selected compounds that contributed more than 1% to the overall chemical proile. 1= C27, 2= 7,16-DiMeC27, 3= 3-meC27, 4= C29, 5= 15-,13-,11-,9-MeC29, 6= 7-MeC29, 7= 11,15-DiMeC29, 8= 9,15 + 9,16-DiMeC29, 9= 7,15 + 7,18-DiMeC29, 10= 3-MeC29, 11= 13,15-DiMeC29, 12= 15-,14-,13-,11-MeC30, 13= 11,15-Di15-,14-,13-,11-MeC30, 14= 15-,13-,11-,9-MeC31, 15= 7-MeC31, 16= 11,16-DiMeC31, 17= 9,21-DiMeC31 and 18= 7,19 + 7,16-DiMeC31.
Asterisks represent identiied contaminants in some of the samples.
N. Cuticular compound DF SS F statistic p-value
1 C27 7 58.57 3.97 < 0.01
2 7,16-dimeC27 7 29.82 0.92 0.49
3 3-meC27 7 29.22 6.54 < 0.001
4 C29 7 33.82 3.55 < 0.01
5 15-,13-,11-,9-meC29 7 6.39 4.30 < 0.001
6 7-meC29 7 17.20 2.85 0.01
7 11,15-dimeC29 7 5.99 0.49 0.83
8 9,15 + 9,16-dimeC29 7 14.11 0.88 0.52 9 7,15 + 7,18-dimeC29 7 93.09 2.46 0.02
10 3-meC29 7 21.03 1.23 0.30
11 13,15-dimeC29 7 75.53 15.07 < 0.001 12 15-,14-,13-,11-meC30 7 33.11 5.42 < 0.001
13 11,15-dimeC30 7 2.63 1.34 0.25
14 15-,13-,11-,9-meC31 7 19.33 2.39 0.03
15 7-meC31 7 15.72 3.80 < 0.01
16 11,16-dimeC31 7 13.09 10.42 < 0.001
17 9,21-dimeC31 7 101.24 2.82 0.01
18 7,19 + 7,16-dimeC31 7 15.07 0.94 0.32 Table 1. Group differences (colony) in total compound quantity for each chemical compound during the post-emergence phase in M. cassununga.
N= number. (ANOVA: p was signiicant for < 0.01 and < 0.001).
ASN Murakami et al - Cuticular Hydrocarbons Proiles and Colonial Recognition in Mischocyttarus cassununga 112
Despite the ability of colonial recognition has been studied for a long time, this aspect was not much investigated in the endemic species from the South America. In the neotropical species Polistes satan, a variation in hydrocarbon proile was suficiently strong to discriminate individuals according to their colony membership. According to this study, it seems that small differences in the proportion of these compounds can be detected and used as a chemical-based cue by nestmates to detect invaders and avoid usurpation (Tannure-Nascimento et al., 2007). The hydrocarbon analysis in M. cassununga shows that the differentiation of the cuticular proile among colonies is related to differences in abundance of a mixture of compounds, especially of branched alkanes. Although we did not perform bioassays in this study, ield observations show that female wasps are aggressive toward non conspeciic individuals. This fact also may indicate that these wasps are capable to use these chemical differences for recognition.
In conclusion, this work in M. cassununga represents a initial investigation of the potential role of cuticular hydrocarbons as mediators of nestmate recognition in the Mischocyttarini tribe. However, more researches are necessary toward a better understanding of the importance of cuticular hydrocarbons in Neotropical social wasps.
Acknowledgments
Thanks to partnership with Faculdade de Ciências Farmacêuticas (Universidade de São Paulo - USP, Ribeirão Preto - SP). Funding was provided by the Capes (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).
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C27 C 5 x C 7
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C29 C 4 x C 5; C 5 x C 8
15-,13-,11-,9-MeC29 C 2 x C 7; C 5 x C 7
13,15-DiMeC29
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15-,14-,13-,11-MeC30 C 2 x C 3; C 2 x C 5; C 3 x C 4; C 4 x C 5
7-MeC31 C 4 x C 5; C 4 x C 7
11,16-DiMeC31
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Table 2. Comparisons between colony groups with signiicant differences according to the cuticular compounds in M. cassununga. C: colony.
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